Regulatory Mechanisms of Sclerostin Expression During Bone Remodeling

Journal of Bone and Mineral Metabolism (2019) 37:9–17 https://doi.org/10.1007/s00774-018-0971-7

INIVITED REVIEW

Regulatory mechanisms of sclerostin expression during bone remodeling

Masanori Koide1 · Yasuhiro Kobayashi1

Received: 12 September 2018 / Accepted: 14 October 2018 / Published online: 24 October 2018 © The Japanese Society for Bone and Mineral Research and Springer Japan KK, part of Springer Nature 2018

Abstract Osteocytes are embedded in bone matrices and are connected to each other to respond to mechanical loading on bone. Recent studies have demonstrated the roles of mechanical loading in bone accrual. Bone responds to mechanical loading by decreasing the expression of sclerostin, an inhibitor of Wnt/β-catenin signals, in osteocytes. This increases bone mass because the activation of Wnt/β-catenin signals in bone microenvironments promotes bone formation and suppresses bone resorption. Thus, in recent years, sclerostin have attracted increasing attention in bone metabolism. However, the regulatory mechanism of sclerostin expression during bone remodeling has not been fully elucidated. In this review, we summarized the regulation of bone formation and resorption by Wnt signals, a Wnt/β-catenin signal inhibitor sclerostin, and molecular mechanisms by which the expression of sclerostin is suppressed by mechanical loading and parathyroid hormone. We also discuss a possibility that osteoclasts suppress the expression of sclerostin during bone remodeling, which in turn, promote bone formation. The efectiveness of an anti-sclerostin antibody with anti-dickkopf-1 antibody for increasing bone mass was discussed.

Keywords Sclerostin · Bone remodeling · Osteocytes · Osteoblasts · Osteoclasts

Introduction osteoclast precursors. Osteoprotegerin (OPG), a decoy receptor of RANKL, is expressed by osteoblasts and osteo- Bone is continuously remodeled throughout life to main- cytes. OPG interferes with the interaction between RANKL tain plasma calcium homeostasis and prevent the accumu- and RANK, which in turn, inhibits osteoclast diferentiation lation of old bone. Osteoclasts resorb bone, and new bone [6, 7]. is then formed by osteoblasts to replace the amount of bone OPG-knockout (KO) mice exhibited increased bone resorbed. This is called bone remodeling, and bone mass resorption due to excess osteoclast diferentiation and activ- is kept constant by balanced bone remodeling due to the ity [8, 9]. In addition, the activity of bone formation mark- coupling between bone resorption and formation [1, 2]. An ers, such as serum alkaline phosphatase, and bone formation imbalance between bone resorption and formation leads to parameters were also increased [8, 10], suggesting that bone either a low bone mass or a high bone mass [1]. formation was also enhanced in OPG-KO mice even though Osteoclasts differentiate from osteoclast precursors, bone mass was decreased. When these mice were treated monocyte–macrophage lineage cells, by stimulation by with the anti-bone-resorbing drug bisphosphonate, bone for- receptor activator of NF-κB ligand (RANKL) and mac- mation was markedly suppressed in association with the sup- rophage colony-stimulating factors (M-CSF) [3–5]. RANKL pression of bone resorption [10]. These results demonstrated and M-CSF bind to receptor activator of NF-κB (RANK) that bone resorption is linked to bone formation by factors and c-fms, respectively. Both receptors are expressed in facilitating the transition from bone resorption to formation, the so-called coupling factors. Several factors have been found to act as coupling factors * Yasuhiro Kobayashi [email protected] between bone resorption and formation [1, 2]. Transforming growth factor beta (TGF-β) and insulin-like growth factor 1 Division of Hard Tissue Research, Institute for Oral Science, I (IGF-I) are embedded in bone matrices during bone for- Matsumoto Dental University, 1780 Gobara, Hiro‑oka, mation [11, 12]. These cytokines are released from bone Shiojiri, Nagano 399‑0781, Japan

Vol.:(0123456789)1 3 10 Journal of Bone and Mineral Metabolism (2019) 37:9–17 matrices during bone resorption and they then induce the migration of osteoblast precursors to bone resorption sites. Osteoclast-derived factors, such as cardiotrophin-1 (CT-1), Wnt10b, sphingosine-1-phosphate, BMP-6, collagen triple- helix repeat-containing 1, and platelet-derived growth factor (PDGF)-BB, were reported to directly act on osteoblast lineage cells, and promote the diferentiation of osteoblasts and bone formation [13–17]. EphrinB2, expressed by osteoclasts, also promotes the differentiation of osteoblasts through EphB4 receptors [18]. Osteocytes reportedly produce several Wnt ligands including Wnt1 [19] and Wnt/β-catenin signal inhibitor sclerostin [20]. The loss-of-function mutations of the SOST gene (encoding sclerostin) cause osteosclerosis with abnormal high bone mass, suggesting that bone formation is usually suppressed by sclerostin through the inhibition of Wnt/β-catenin signals to achieve normal bone development. Fig. 1 Wnt signals. Wnt ligands activate β-catenin-dependent canoni- However, it is not fully examined whether osteoclast-derived cal and -independent non-canonical signals. Wntless, an 8-pass factors also act on osteocytes to inhibit the expression of transmembrane protein, is necessary for Wnt secretion. Wntless-def- sclerostin, which in turn, promote bone formation. cient cells failed to secrete all Wnt ligands. Wnt ligands bind to the receptor complex of frizzled and LRP5/6, and inhibit the activity of In this review, we introduced roles of Wnt signals in bone β-catenin destruction complexes. This leads to cytosolic accumulation formation and regulatory mechanism of sclerostin expres- and nuclear translocation of β-catenin. Nuclear β-catenin together sion by PTH. We would like to propose a possibility that with TCF/LEF induces the transcription of the target genes. Scle- osteoclasts suppress the expression of sclerostin during bone rostin and Dkk-1 bind to LRP5/6, and inhibit Wnt/β-catenin signals. Binding of sclerostin to LRP4 is required for the inhibitory action of remodeling, which in turn, promote bone formation [21]. In sclerostin. Wnt ligands such as Wnt5a binds receptor complex of friz- addition, we discussed the efects of anti-Dkk-1 and anti- zled and Ror1/2, which in turn activates β-catenin-independent non- sclerostin antibodies on bone accrual. canonical signals

Regulation of bone formation by Wnt/ also increased in DA-β-catenin Ocy mice, thereby increas- β‑catenin signals ing bone resorption. These fndings suggest that osteocytes as well as osteoblasts play a critical role in bone anabolic Wnt ligands activate β-catenin-dependent canonical and action of Wnt/β-catenin signals although the mechanism -independent non-canonical signaling pathways [22] by which the expression of RANKL was increased in (Fig. 1). Canonical and non-canonical Wnt signals regu- DA-β-catenin Ocy mice remains to be elucidated. late bone formation and resorption [23]. At present, 19 Wnt Non-canonical Wnt signals impact bone resorption. ligands have been identifed in humans and mice [22]. Wnt Non-canonical Wnt5a binds to its receptor Ror2 in osteo- ligands, such as Wnt1, Wnt5a, Wnt7b, and Wnt10b, report- clast precursors and promotes the expression of RANK, edly regulate bone formation [19, 24–27]. which in turn increased RANKL-induced osteoclast Analysis of mice having gain-of-function and loss- formation [24]. Wnt5a-Ror2 signals also promote bone- of-function mutations of β-catenin in mature osteoblasts resorbing activity of osteoclasts through small GTPase revealed that Wnt/β-catenin signals markedly increase Rho-Pkn3-c-Src signaling axis [30]. In contrast to Wnt5a, bone mass due to the decreased bone resorption without Wnt16 reportedly activated canonical Wnt signals in oste- afecting bone formation [28]. This study has shown that oblastic cells and non-canonical signals in osteoclast pre- activation of Wnt/β-catenin signals in mature osteoblasts cursors [31]. Wnt16-defcient mice exhibited low cortical promotes OPG expression, thereby inhibiting osteoclas- but not trabecular bone mass [31]. Osteoblastogenesis is togenesis. Ten years later, mice expressing dominant normal in those Wnt16-defcient calvaria-derived osteo- active form of β-catenin in osteocytes (DA-β-catenin Ocy blasts in cultures. Treatment of osteoblastic MC3T3-E1 mice) reportedly exhibited high bone mass associated with cells with Wnt16 increased the expression of OPG through increased bone resorption and formation [29]. Similar to the Wnt/β-catenin signals. Furthermore, Wnt16 inhibited mice expressing constitutively active form of β-catenin RANKL-induced osteoclast formation by suppression of in mature osteoblasts [28], the expression of OPG was NF-κB and NFATc1 signals [31]. These results suggest increased in those mice. The expression of RANKL was that Wnt16 inhibits RANK signals in osteoclast precursors

1 3 Journal of Bone and Mineral Metabolism (2019) 37:9–17 11 and induces the expression of OPG in osteoblasts, which bone mass [44, 45]. These fndings suggest that sclerostin in turn, inhibits osteoclast formation. inhibits Wnt/β-catenin signals and suppresses bone forma- Wntless (Wls), an 8-pass transmembrane protein, is tion (Fig. 1). required for the secretion of Wnt ligands [32, 33] (Fig. 1). LRP4 is reportedly required for the inhibitory action of Therefore, Wnt ligands cannot be secreted from Wls-def- sclerostin (46). Two mutations in the LRP4 gene (R1170W cient cells. Both canonical and non-canonical Wnt signals and W1186S) have been found in patients with sclerosteo- are inactivated in those cells. As several kinds of Wnt sis [46]. Sclerostin failed to bind these LRP4 mutants and ligands are secreted from osteoblasts, Wls-defcient osteo- inhibit Wnt/β-catenin signals in cells expressing these blasts are useful to analyze the roles of osteoblast-derived mutants. These fndings indicated that LRP4 bound to scle- Wnt ligands in bone formation. Osteoblast-specifc Wls-con- rostin to facilitate the inhibitory action of sclerostin. Osteo- ditional knockout (cKO) mice exhibit a marked decrease in blast-specifc Lrp4 cKO mice (Lrp4fox/fox; osteocalcin-cre trabecular bone and cortical bone [34]. Histomorphometric mice) exhibited a high bone mass with increased bone for- analysis revealed a signifcant reduction of the bone forma- mation [47, 48]. These fndings strongly indicate that LRP4 tion rate in Wls-cKO mice [34]. Furthermore, the expres- facilitates the inhibitory action of sclerostin in vivo. sion of osteoblast marker genes, such as Runx2 and Osterix, Dickkopf-1 (Dkk-1) is also an inhibitor of Wnt/β-catenin as well as the Wnt target gene Axin2 was significantly signals. Dkk-1 binds to LRP5/6 receptors and a transmem- decreased in Wls-defcient osteoblasts [34]. Mineralization brane protein kremen, and the complex of Dkk-1, kremen, was also decreased in these cells. These fndings suggested and Lrp5/6 internalizes from cell surfaces, thereby inhibit- that Wnt ligands secreted from osteoblast lineage cells are ing Wnt/β-catenin signals [49]. Dkk-1 is highly expressed important for osteoblast diferentiation and bone formation. in bone tissue. Mice having heterozygous deletion of the Low-density lipoprotein receptor-related protein (LRP) Dkk-1 gene exhibited increased bone mass [50], whereas 5/6 functions as a co-receptor for Wnt/β-catenin signals. Dkk-1 transgenic mice exhibited decreased bone mass [51]. Analysis of loss-of-function and gain-of-function muta- Bone resorption markers such as urine deoxypyridinoline tions in the Lrp5 gene demonstrated that Wnt/β-catenin sig- and the number of osteoclasts remained unchanged in those nals promote osteoblast diferentiation and increase bone mice, suggesting that Dkk-1 is not strongly involved in mass [35]. Furthermore, Osterix failed to be expressed by osteoclast formation during developmental process [50]. β-catenin-defcient perichondrial cells or periosteal cells [36, Furthermore, administration of an anti-Dkk-1 neutralizing 37]. Thus, Wnt/β-catenin signals are necessary for osteoblast antibody reportedly increased bone mass by promoting bone diferentiation and bone formation. formation [52]. These fndings demonstrate that Wnt/β- catenin signals in bone are tightly regulated by Dkk-1 and bone-specifc sclerostin. Roles of Wnt inhibitors in bone formation

The SOST gene is a responsible gene for osteosclero- Efects of mechanical loading sis and van Buchem disease. A loss-of-function mutation on the sclerostin expression in the SOST gene causes sclerosteosis with an abnormal increase in bone mass [38, 39]. A homozygous 52-kb non- Osteocytes sense changes in bone fluid flow caused by coding deletion (ECR5 region) of the SOST gene causes Van mechanical loading, and are involved in the increase and Buchem disease, which presents the same clinical features maintenance of bone mass [53]. Loading in upper limbs as sclerosteosis [40]. The ECR5 region in the SOST gene is reduced the expression of sclerostin and Dkk-1 in ulna- a bone-specifc enhancer of sclerostin expression in osteo- loading model mice [54]. In contrast, unloading increased cytes [41]. Sclerostin, a glycoprotein secreted from osteo- the expression of sclerostin in the lower limbs of tail-sus- cytes, binds to the frst two YWTD-EGF repeat domains pended mice and then decreased bone formation [55]. Bone of LRP5/6 and inhibits Wnt/β-catenin signals [42]. Similar formation failed to decrease in the unloaded lower limbs of to sclerosteosis, Sost-KO mice exhibited high bone mass Sost-KO mice [55]. Furthermore, transgenic mice express- with a marked increase in bone formation [43]. Bone resorp- ing human sclerostin in osteocytes exhibited the suppres- tion parameters such as serum TRAP5b and the number of sion of mechanical loading-induced bone formation [56]. osteoclasts remained unchanged, indicating that the def- These fndings revealed that mechanical loading suppresses ciency of sclerostin does not afect osteoclast formation. the expression of sclerostin in osteocytes, which in turn, This fnding suggests that sclerostin is not strongly involved promotes bone formation. in osteoclast formation during developmental process. Fur- Periostin is a secreted extracellular matrix protein thermore, administration of anti-sclerostin neutralizing anti- expressed in the periosteum and periodontal ligaments [57]. body reportedly promoted bone formation and increased Periostin (encoded by the Postn gene)-KO mice developed

1 3 12 Journal of Bone and Mineral Metabolism (2019) 37:9–17 osteoporosis and periodontitis [58]. Mechanical loading also reportedly induced the expression of periostin and suppressed sclerostin expression [59]. Bonnet et al. [59, 60] analyzed the expression of sclerostin in osteocytes and the bone mass of cortical bone in Postn-KO mice treated with PTH or mechanical loading. In Postn-KO mice, administration of PTH or mechanical loading failed to decrease the expression of sclerostin, and the bone mass in cortical bone was not increased. In contrast, administration of an anti-sclerostin neutralizing antibody increased the bone mass in cortical bone of Postn-KO mice. Therefore, administration of PTH or mechanical loading increased the expression of periostin in the periosteum, and the increased periostin reduced the expression of sclerostin, which in turn, promoted bone formation in cortical bone (Fig. 2). These results suggest a new function of periostin as a suppressor of sclerostin expression. Fig. 3 PTH signals suppress the expression of sclerostin. MEF2 binds The mechanisms by which periostin suppresses the expres- the ECR5 (enhancer region) of the Sost gene to induce the expression sion of sclerostin need to be clarifed in the future. of sclerostin. Salt-induced kinases (SIKs) phosphorylate HDAC4/5 to promote complex formation of HDAC4/5 and 14-3-3, which in turn retains HDAC4/5 in the cytoplasm. PTH signals inhibit the kinase activity of SIKs, which in turn increases dephosphorylated HDAC Regulatory mechanism of sclerostin in nucleus. Nuclear dephosphorylated HDAC inhibits the activity of expression by PTH and IL‑6 family cytokines MEF2

As described above, the down-regulation of sclerostin of MEF2 to the ECR5 region and decreased the expression expression is considered to be an important step for bone of sclerostin [65]. accrual. Because the efect of PTH on the expression of scle- The detailed roles of MEF2 in the expression of sclerostin is well-studied, here we would like to introduce the rostin have been proposed. Histone deacetylase (HDAC) 5 inhibitory action of PTH in sclerostin expression. Intermit- binds to MEF2 and suppresses the expression of sclerostin tent administration of PTH increases bone mass [61, 62]. [66] (Fig. 3). The expression of sclerostin was enhanced As PTH markedly suppresses the expression of sclerostin in HDAC5-KO mice, which exhibited decreased bone in osteocytes, the role of PTH in bone accrual is consid- formation [66], suggesting that HDAC5 suppresses the ered to be mediated, in part, by the inhibition of sclerostin expression of sclerostin. Knockdown of HDAC5 enhanced expression [63, 64]. The 52-kb region (ECR5) containing the transcriptional activity of MEF2 and the expression the enhancer downstream of the Sost gene is important for of sclerostin. Taken together, the association of HDAC5 bone-specifc sclerostin expression [40] (Fig. 3). Myocyte with MEF2 in osteocytes reduced the transcription activ- enhancer factor (MEF) 2, a transcription factor, binds to the ity of MEF2, and subsequently the expression of sclerostin. ECR5 region and induces the expression of sclerostin [65]. Recently, a further detailed mechanism was reported. Salt- Stimulation of PTH in osteocytes reduced the recruitment inducible kinases (SIKs), which are a kinase for HDAC4/5 and cAMP-regulated transcriptional coactivators (CRTC) [67], were found to phosphorylate HDAC5 to promote the complex formation of HDAC5 with 14-3-3 proteins. 14-3-3 proteins are intracellular dimeric proteins involved in vari- ous biological events such as signal transductions. 14-3-3 retained HDAC5 in the cytoplasm [68, 69]. Stimulation of PTH suppressed the activity of SIKs and increased dephosphorylated HDAC4/5, which localizes to the nucleus. The nuclear HDAC4/5 forms a complex with MEF2 and inhibits the transcriptional activity of MEF2, which in turn, suppresses the expression of sclerostin [69] (Fig. 3). Further- more, SIK inhibitors have been reported to promote bone formation. Of note, the suppression of SIK activity was Fig. 2 Roles of periostin in the suppression of sclerostin expression. Periostin secreted from periosteal osteoblasts acts on osteocytes and demonstrated to increase the expression of RANKL via inhibits the expression of sclerostin nuclear translocation of dephosphorylated CRTC 2 [69].

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This suggests that SIK is also involved in osteoclastogenesis oncostatin M suppresses the expression of sclerostin and when osteoblast linage cells are stimulated by PTH. In the increases that of RANKL to induce osteoclast formation future, clarifcation as to whether SIKs and HDAC4/5 are during bone remodeling. They also suggest that osteoclast- involved in the mechanical loading-induced suppression of derived CT-1 also suppresses the expression of sclerostin. sclerostin expression is needed. However, it is unknown how CT-1 regulates the expression Roles of PTH1 receptor (PTH1R) in osteocytes in the of sclerostin in vivo. expression of sclerostin have been examined using dentin matrix protein (DMP)-1-Cre: PTH1R conditional knockout mice. Powell et al. [70] prepared tamoxifen-inducible Roles of sclerostin in bone remodeling PTH1R cKO mice using DMP-1-CreERT2 mice. These PTH1R cKO mice exhibited low bone mass with the During bone remodeling, several coupling factors are con- increased expression of sclerostin mRNA. The expression sidered to link bone resorption with formation because of Wnt target gene axin was decreased in bone tissues from bone resorption promotes bone formation [1, 2]. Excellent those mice. These results suggest that PTH1R signals sup- review articles describe roles of osteocytes in bone remod- press the expression of sclerostin, which in turn, promotes eling [73–75]. Osteocytes response to mechanical loading bone accrual under physiological condition. In contrast to and PTH, and then decrease the expression of sclerostin, tamoxifen-inducible PTH1R cKO mice [70], PTH1R cKO thereby promoting bone formation. Osteocytes also produce mice using non-inducible DMP-1 Cre exhibited high bone RANKL to induce bone resorption. Thus, osteocytes regu- mass with a modest decrease in bone resorption. These late bone remodeling. results suggest that PTH1R signal in osteocytes may not As mentioned above, OPG-KO mice exhibited high bone be involved in the suppression of sclerostin expression by turnover with increased bone resorption and formation. The PTH [71]. However, the expression of Wnt target genes such expression of sclerostin was reportedly lower in OPG-KO as Naked2 and Conexin 43 were decreased in those mice, mice [76]. However, the mechanisms have not been fully suggesting that Wnt/β-catenin signals suppressed probably elucidated at the molecular level. We analyzed OPG-KO due to the increase in the expression of sclerostin. Further- mice and found that osteoclasts promoted bone formation more, the anabolic action and the suppression of sclerostin via the suppression of sclerostin expression in osteocytes expression by intermittent injections of PTH disappeared in [21]. Antibody array analysis and real-time PCR analysis those cKO mice [71], suggesting that PTH1R signals play demonstrated that mature osteoclasts abundantly expressed an important role in anabolic action of PTH by the sup- and secreted LIF [21]. LIF secreted from osteoclasts pression of sclerostin expression. Those cKO mice exhibited reduced the expression of sclerostin in osteocytes and cul- the decreased expression of RANKL and the decrease in tured UMR106 cells [21]. Anti-LIF antibody, but not anti- bone resorption marker serum CTX. These fndings suggest oncostatin M antibody, neutralized the suppressive efects of that bone mass is increased due to the suppression of bone osteoclast-conditioned medium on sclerostin expression in resorption in those cKO mice. The increased bone mass in UMR106 cells [21], suggesting that osteoclast-conditioned those cKO mice might be due to cre recombinase activity medium contains enough LIF to suppress the expression of during developmental process. Further studies are needed sclerostin. Furthermore, administration of the anti-RANKL to clarify this point. antibody inhibited osteoclast diferentiation and inhibited Recently, we found that osteoclast-derived leukemia bone resorption in OPG-KO mice, and increased the expres- inhibitory factor (LIF) suppresses the expression of scle- sion of sclerostin by osteocytes [21]. Wnt/β-catenin signals rostin in UMR106 cells [21]. Therefore, we introduce efects in bone tissues were decreased in mice treated with the anti- of IL-6 family cytokines such as LIF, oncostatin M, and RANKL antibody, which in turn, suppressed bone forma- CT-1 on the expression of sclerostin. These IL-6 family tion [21]. These results suggest that regulation of sclerostin members reportedly suppress the expression of sclerostin expression by osteoclasts plays an important role in the in cultured UMR106 cells [72]. Oncostatin M was reported coupling between bone resorption and formation (Fig. 4). to be abundantly expressed in osteoblasts [72]. Furthermore, A clinical study found that the administration of the anti- administration of recombinant oncostatin M decreased the RANKL antibody reduced bone formation markers associ- expression of sclerostin in wild-type mice [72]. IL-6 family ated with the suppression of bone resorption markers [77, cytokines such as IL-6 and oncostatin M reportedly induced 78]. Moreover, administration of the anti-RANKL antibody the expression of RANKL to promote osteoclast formation increased the amount of sclerostin in serum [79, 80]. These in co-cultures of bone marrow cells with osteoblastic cells clinical studies strongly suggested that bone resorption sup- [5]. The treatment with oncostatin M also increased the presses the expression of sclerostin, which in turn, promotes expression of RANKL in osteoblastic cell line KUSA 4b10 bone formation in humans. Thus, simultaneous administra- cells [72]. These results indicate that osteoblast-derived tion of anti-sclerostin antibodies with RANKL antibodies

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of sclerostin. In contrast, treatment of ovariectomized rats with the anti-sclerostin antibody increased the expression of Dkk-1[83]. Administration of both anti-Dkk-1 and anti-sclerostin antibodies more markedly increased the bone volume than only Dkk-1 antibody or sclerostin antibody in fracture model rats [83]. In addition, administration of a bispecifc antibody targeting sclerostin and Dkk-1 promoted fracture healing as compared with the anti-sclerostin antibody [84]. These results suggest that the inhibition of both sclerostin and Dkk-1 is useful for promoting bone formation compared with neutralizing either sclerostin or Dkk-1, and that the inhibition of sclerostin induces the expression of Dkk-1 Fig. 4 LIF secreted from osteoclasts inhibits the expression of scle- because Dkk-1 is a target gene of Wnt/β-catenin signals. rostin in osteocytes. Osteocytes produce sclerostin, which inhibits Wnt/β-catenin signals in osteoblasts and suppresses excess bone formation. Osteoclasts secrete LIF (1) and inhibit the expression of sclerostin (2), which in turn, promotes bone formation through the activation of Wnt/β-catenin signals (3). Osteocytes are involved in the Conclusion coupling between bone resorption and formation In this review, we introduced the regulatory mechanism of maybe efective for keeping bone formation rate, because sclerostin expression, roles of osteocytes in the coupling administration of anti-RANKL antibodies suppresses bone between bone resorption and formation, and the useful- formation due to up-regulation of sclerostin expression. It is ness of sclerostin and Dkk-1 inhibition for bone accrual also important that the regulatory mechanisms of sclerostin and fracture treatment. Osteoclasts secrete LIF and inhibit expression by bone resorption be elucidated in more detail the expression of sclerostin, which in turn, promote bone in the future. formation through the activation of Wnt/β-catenin signals. Although recent studies established important roles of sclerostin in bone metabolism, several molecular mechanisms, Anti‑sclerostin antibody and anti‑DKK1 such as the suppression of sclerostin expression by mechani- antibody cal loading and LIF, remain to be elucidated. These issues will be solved in future studies, leading to the development Recently, Witcher et al. [81] reported that conditional dele- of drugs for osteoporosis. tion of the Dkk-1 gene using DMP-1 Cre and treatment of Acknowledgements This work was supported in part by Grants-in-Aid wild-type mice with an anti-Dkk-1 antibody had negligible 18H02980 (M.K.), 16H02691 (Y. K.), 18H05388 (Y. K.) from the Min- efects on bone accrual. Administration of the anti-Dkk-1 istry of Education, Culture, Sports, Science and Technology of Japan. antibody increased the expression of sclerostin [81], suggesting that it is regulated by Wnt/β-catenin signals. This Compliance with ethical standards fnding is consistent with the previous fnding that genetic activation of β-catenin in osteocytes increases the expression Conflict of interest The authors declare that they have no confict of of sclerostin in bone [82]. Thus, Wnt/β-catenin signals posi- interest. tively regulate the expression of sclerostin. Further studies are needed to clarify whether Wnt/β-catenin signals regulate the transcription activity of the Sost gene. Administration of References the anti-Dkk-1 antibody to Sost-KO mice further enhanced bone formation even though Sost-KO mice exhibited high 1. Sims NA, Martin TJ (2014) Coupling the activities of bone for- bone mass. The combined administration of anti-DKK-1 and mation and resorption: a multitude of signals within the basic anti- sclerostin antibodies markedly promoted bone forma- multicellular unit. Bonekey Rep 3:481 tion. These results suggest that the increased expression of 2. Crane JL, Cao X (2014) Bone marrow mesenchymal stem cells and TGF-beta signaling in bone remodeling. J Clin Invest sclerostin by inhibition of Dkk-1 weakens the stimulatory 124:466–472 efects of the anti-Dkk-1 antibody on bone formation. In 3. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kinosaki M, addition, postnatal global deletion of the Dkk-1 gene report- Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda edly caused high bone mass [83]. Of note, the expression E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T (1998) Osteoclast diferentiation factor is a ligand for osteopro- of sclerostin was higher in these mice [83]. This also sug- tegerin/osteoclastogenesis-inhibitory factor and is identical to gests that Wnt/β-catenin signals promote the expression TRANCE/RANKL. Proc Natl Acad Sci USA 95:3597–3602

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